The response of cells of monocytic lineage (MO) to bacterial lipopolysaccharide (endotoxin, LPS) is a central event in initiating septic shock during gram-negative sepsis and endotoxemia and in the development of antibody independent tolerance to LPS. The long term goal of this research is to understand the molecular mechanisms that control recognition of LPS by MO. Recent discoveries from our laboratory have established a unique, new model to explain how MO recognize LPS under physiologic conditions. In blood LPS forms a high affinity complex with a plasma protein we discovered; lipopolysaccharide binding protein (LBP) and the LPS-LBP complex binds to the plasma membrane of MO via a receptor that we have identified as CD14, a MO membrane antigen of previously unknown function. The net effects of utilizing the LBP/CD14 pathway are to markedly enhance MO sensitivity to LPS and to induce more rapid responses, i.e. cytokine release, compared to responses to LPS in the absence of the LBP/CD14 pathway. Bearing in mind that MO respond to LPS independently of the LBP/CD14 pathway we propose experiments to test the following hypothesis explaining how LBP and CD14 act to facilitate MO responses to LPS. Engagement of CD14 by LPS-LBP complexes results in a biochemical signal that facilitates binding of LPS to an additional membrane protein, the putative lipid A receptor. It is this event that links the LBP/CD14 dependent and independent pathways of LPS-induced MO stimulation. The SPECIFIC AIMS of this proposal combine immunocytochemical and biochemical approaches to define the initial steps involved in MO recognition of LPS via the LBP/CD14 dependent pathway and events that regulate these steps. Cross-linking strategies will be used to identify, purify and ultimately clone the putative lipid A receptor. These studies will also provide a unique framework to better understand the biochemical mechanisms in MO that lead to LPS-induced tolerance to its own effects. The proposed studies will significantly impact our understanding of pathogenic mechanisms in human diseases such as gram-negative septic shock and ARDS. Implicit in the basic studies proposed here is the development of new therapeutic strategies to intervene in these diseases.